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Antonie Van Leeuwenhoek Nov 2022Analysis of predicted fungal proteomes revealed a large family of sequences that showed similarity to the Saccharomyces cerevisiae Class-I dihydroorotate dehydrogenase...
Analysis of predicted fungal proteomes revealed a large family of sequences that showed similarity to the Saccharomyces cerevisiae Class-I dihydroorotate dehydrogenase Ura1, which supports synthesis of pyrimidines under aerobic and anaerobic conditions. However, expression of codon-optimised representatives of this gene family, from the ascomycete Alternaria alternata and the basidiomycete Schizophyllum commune, only supported growth of an S. cerevisiae ura1Δ mutant when synthetic media were supplemented with dihydrouracil. A hypothesis that these genes encode NAD(P)-dependent dihydrouracil dehydrogenases (EC 1.3.1.1 or 1.3.1.2) was rejected based on absence of complementation in anaerobic cultures. Uracil- and thymine-dependent oxygen consumption and hydrogen-peroxide production by cell extracts of S. cerevisiae strains expressing the A. alternata and S. commune genes showed that, instead, they encode active dihydrouracil oxidases (DHO, EC1.3.3.7). DHO catalyses the reaction dihydrouracil + O → uracil + HO and was only reported in the yeast Rhodotorula glutinis (Owaki in J Ferment Technol 64:205-210, 1986). No structural gene for DHO was previously identified. DHO-expressing strains were highly sensitive to 5-fluorodihydrouracil (5F-dhu) and plasmids bearing expression cassettes for DHO were readily lost during growth on 5F-dhu-containing media. These results show the potential applicability of fungal DHO genes as counter-selectable marker genes for genetic modification of S. cerevisiae and other organisms that lack a native DHO. Further research should explore the physiological significance of this enigmatic and apparently widespread fungal enzyme.
Topics: Saccharomyces cerevisiae; Hydrogen Peroxide; Thymine; Proteome; Cell Extracts; NAD; Genes, Fungal; Uracil; Hydrogen
PubMed: 36241945
DOI: 10.1007/s10482-022-01779-9 -
Microbial Cell Factories Aug 2022The regioselective hydroxylation of phenolic compounds, especially flavonoids, is still a bottleneck of classical organic chemistry that could be solved using enzymes...
BACKGROUND
The regioselective hydroxylation of phenolic compounds, especially flavonoids, is still a bottleneck of classical organic chemistry that could be solved using enzymes with high activity and specificity. Yeast Rhodotorula glutinis KCh735 in known to catalyze the C-8 hydroxylation of flavones and flavanones. The enzyme F8H (flavonoid C8-hydroxylase) is involved in the reaction, but the specific gene has not yet been identified. In this work, we present identification, heterologous expression and characterization of the first F8H ortho-hydroxylase from yeast.
RESULTS
Differential transcriptome analysis and homology to bacterial monooxygenases, including also a FAD-dependent motif and a GD motif characteristic for flavin-dependent monooxygenases, provided a set of coding sequences among which RgF8H was identified. Phylogenetic analysis suggests that RgF8H is a member of the flavin monooxygenase group active on flavonoid substrates. Analysis of recombinant protein showed that the enzyme catalyzes the C8-hydroxylation of naringenin, hesperetin, eriodyctiol, pinocembrin, apigenin, luteolin, chrysin, diosmetin and 7,4'-dihydroxyflavone. The presence of the C7-OH group is necessary for enzymatic activity indicating ortho-hydroxylation mechanism. The enzyme requires the NADPH coenzyme for regeneration prosthetic group, displays very low hydroxyperoxyflavin decupling rate, and addition of FAD significantly increases its activity.
CONCLUSIONS
This study presents identification of the first yeast hydroxylase responsible for regioselective C8-hydroxylation of flavonoids (F8H). The enzyme was biochemically characterized and applied in in vitro cascade with Bacillus megaterium glucose dehydrogenase reactions. High in vivo activity in Escherichia coli enable further synthetic biology application towards production of rare highly antioxidant compounds.
Topics: Flavin-Adenine Dinucleotide; Flavins; Flavonoids; Hydroxylation; Mixed Function Oxygenases; Phylogeny; Rhodotorula; Substrate Specificity
PubMed: 36038906
DOI: 10.1186/s12934-022-01899-x -
Journal of Fungi (Basel, Switzerland) Jun 2022Exploration of yeast diversity for the sustainable production of biofuels, in particular biodiesel, is gaining momentum in recent years. However, sustainable, and... (Review)
Review
Exploration of yeast diversity for the sustainable production of biofuels, in particular biodiesel, is gaining momentum in recent years. However, sustainable, and economically viable bioprocesses require yeast strains exhibiting: (i) high tolerance to multiple bioprocess-related stresses, including the various chemical inhibitors present in hydrolysates from lignocellulosic biomass and residues; (ii) the ability to efficiently consume all the major carbon sources present; (iii) the capacity to produce lipids with adequate composition in high yields. More than 160 non-conventional (non-) yeast species are described as oleaginous, but only a smaller group are relatively well characterised, including , , , , and . This article provides an overview of lipid production by oleaginous yeasts focusing on yeast diversity, metabolism, and other microbiological issues related to the toxicity and tolerance to multiple challenging stresses limiting bioprocess performance. This is essential knowledge to better understand and guide the rational improvement of yeast performance either by genetic manipulation or by exploring yeast physiology and optimal process conditions. Examples gathered from the literature showing the potential of different oleaginous yeasts/process conditions to produce oils for biodiesel from agro-forestry and industrial organic residues are provided.
PubMed: 35887443
DOI: 10.3390/jof8070687 -
Brazilian Journal of Microbiology :... Sep 2022This work aimed to characterize the antimicrobial compounds obtained from the potential probiotic Lactiplantibacillus plantarum S61, isolated from traditional fermented...
This work aimed to characterize the antimicrobial compounds obtained from the potential probiotic Lactiplantibacillus plantarum S61, isolated from traditional fermented green olive, involved in their activity against fungi and bacteria responsible for food spoilage and poisonings. Their application as a biopreservative agent was also investigated. The culture of L. plantarum S61 showed substantial antifungal and antibacterial activity against yeasts (Rhodotorula glutinis and Candida pelliculosa), molds (Penicillium digitatum, Aspergillus niger, Fusarium oxysporum, and Rhizopus oryzae), and pathogenic bacteria (Listeria monocytogenes ATCC 19,117, Salmonella enterica subsp. enterica ATCC 14,028, Staphylococcus aureus subsp. aureus ATCC 6538, Pseudomonas aeruginosa ATCC 49,189), with inhibition zones > 10 mm. Likewise, the cell-free supernatant (CFS) of L. plantarum S61 showed an essential inhibitory effect against fungi and bacteria, with inhibition diameters of 12.25-22.05 mm and 16.95-17.25 mm, respectively. The CFS inhibited molds' biomass and mycelium growth, with inhibition ranges of 63.18-83.64% and 22.57-38.93%, respectively. The antifungal activity of the CFS was stable during 4 weeks of storage at 25 °C, while it gradually decreased during storage at 4 °C. Several antimicrobial compounds were evidenced in the CFS of L. plantarum S61, including organic acids, ethanol, hydrogen peroxide, diacetyl, proteins, and fatty acids. The protein fraction, purified by reversed-phase high-performance liquid chromatography (RP-HPLC), demonstrated important antifungal activity, in relation to the fraction with molecular weight between 2 and 6 kDa. L. plantarum S61 and its CFS, tested in apple and orange fruit biopreservation, demonstrated their protective effect against P. digitatum spoilage. The CFS exhibited effectiveness in reducing Salmonella enterica subsp. enterica ATCC 14,028 in apple juice. L. plantarum S61 and/or its bioactive compounds CFS represent a promising strategy for biocontrol against pathogens and spoilage microorganisms in the agro-industry.
Topics: Anti-Bacterial Agents; Anti-Infective Agents; Antifungal Agents; Fungi; Lactobacillus plantarum; Listeria monocytogenes; Probiotics; Salmonella
PubMed: 35804284
DOI: 10.1007/s42770-022-00791-5 -
Frontiers in Nutrition 2022, as a member of the family , is of great value in the field of biotechnology. However, the evolutionary relationship of X-20 with , , and are not well understood, and...
, as a member of the family , is of great value in the field of biotechnology. However, the evolutionary relationship of X-20 with , , and are not well understood, and its metabolic pathways such as carotenoid biosynthesis are not well resolved. Here, genome sequencing and comparative genome techniques were employed to improve the understanding of X-20. Phytoene desaturase (crtI) and 15-cis-phytoene synthase/lycopene beta-cyclase (crtYB), key enzymes in carotenoid pathway from X-20 were more efficiently expressed in INVSc1 than in CEN.PK2-1C. High yielding engineered strains were obtained by using synthetic biology technology constructing carotenoid pathway in and optimizing the precursor supply after fed-batch fermentation with palmitic acid supplementation. Genome sequencing analysis and metabolite identification has enhanced the understanding of evolutionary relationships and metabolic pathways in X-20, while heterologous construction of carotenoid pathway has facilitated its industrial application.
PubMed: 35782944
DOI: 10.3389/fnut.2022.918240 -
Microorganisms Jun 2022This work reports the effect of simple feeding strategies and temperature to obtain high-cell-density cultures of Rhodotorula glutinis var. rubescens LOCKR13 maximizing...
This work reports the effect of simple feeding strategies and temperature to obtain high-cell-density cultures of Rhodotorula glutinis var. rubescens LOCKR13 maximizing the de novo lipid productivity using deproteinated potato wastewater (DPW) as a basic medium. Feeding DPW with glucose enables a high yield of Rhodotorula glutinis var. rubescens LOCKR13 biomass (52 g d.w. L−1) to be obtained. The highest values of lipid accumulation (34.15%, w/w), production (14.68 g L−1) and yield coefficients (YL/S: 0.242 g g−1), and volumetric productivity (PL: 0.1 g L−1 h−1) were reached by the strain in the two-stage fed-batch process at 20 °C. The lipid of yeast biomass was rich in oleic acid (Δ9C18:1) and palmitic acid (C16:0), and the lower temperature of incubation significantly increased the MUFA (especially oleic acid) content. For the first time, a unique set of thermal analyses of the microbial oil was performed. The isotherms of the oxidation kinetics (PDSC) showed that lipids extracted from the biomass of red yeast had high oxidative stability. This feature of the yeast oil can be useful for long-shelf-life food products and can be promising for the production of biodiesel.
PubMed: 35744771
DOI: 10.3390/microorganisms10061253 -
Maedica Mar 2022is a widely distributed fungus that has evolved as an important pathogen, especially in immunocompromised individuals, causing fungemias, central nervous system...
is a widely distributed fungus that has evolved as an important pathogen, especially in immunocompromised individuals, causing fungemias, central nervous system infections, peritoneal dialysis-associated peritonitis and keratitis. Even though there are a few reports of skin and soft tissue infections caused by , all these cases involve immunocompromised individuals. is a Gram-positive bacterium known to cause several infections such as bloodstream, urinary tract and skin and soft tissue infections. We report a mixed and skin and soft tissue infection in a 63-year-old woman with well controlled type 2 diabetes mellitus and no other known history of immunosuppression, suffering from skin and soft tissue infection of the right lower extremity. The patient did not respond to treatment with broad spectrum antimicrobials, but had a successful outcome with fluconazole, after was isolated from pus of the skin ulcer. The antifungal treatment led to eradication of the infection, while no recurrence was observed during a follow-up period of two years. Clinicians should be aware that can cause infection even in immunocompetent patients.
PubMed: 35733763
DOI: 10.26574/maedica.2022.17.1.211 -
Microbial Cell Factories Dec 2021Oleaginous yeasts are promising microbial platforms for sustainable, bio-based production of biofuels and oleochemical building blocks. Bio-based residues provide...
BACKGROUND
Oleaginous yeasts are promising microbial platforms for sustainable, bio-based production of biofuels and oleochemical building blocks. Bio-based residues provide sustainable and cost-effective carbon sources for fermentative yeast oil production without land-use change. Considering the regional abundancy of different waste streams, we chose complex biomass residue streams of marine origin; macroalgae hydrolysate, and terrestrial origin; wheat straw hydrolysate in the presence, and absence of corn steep liquor as a complex nitrogen source. We investigated the biomass and lipid yields of an array of well-described oleaginous yeasts; R. glutinis, T. asahii, R. mucilaginosa, R. toruloides, C. oleaginosus growing on these hydrolysates. Furthermore, their sugar utilization, fatty acid profile, and inhibitory effect of the hydrolysates on yeast growth were compared. For correlative reference, we initially performed comparative growth experiments for the strains on individual monomeric sugars separately. Each of these monomeric sugars was a dominant carbon source in the complex biomass hydrolysates evaluated in this study. In addition, we evaluated N-acetylglucosamine, the monomeric building block of chitin, as a low-cost nitrogen and carbon source in yeast fermentation.
RESULTS
C. oleaginosus provided the highest biomass and lipid yields. In the wheat straw and brown algae hydrolysates, this yeast strain gained 7.5 g/L and 3.8 g/L lipids, respectively. Cultivation in algae hydrolysate resulted in a higher level of unsaturated fatty acids in the lipids accumulated by all yeast strains. R. toruloides and C. oleaginosus were able to effectively co-utilize mannitol, glucose, and xylose. Growth rates on wheat straw hydrolysate were enhanced in presence of corn steep liquor.
CONCLUSIONS
Among the yeast strains investigated in this study, C. oleaginosus proved to be the most versatile strain in terms of substrate utilization, productivity, and tolerance in the complex media. Various fatty acid profiles obtained on each substrate encourage the manipulation of culture conditions to achieve the desired fatty acid composition for each application. This could be accomplished by combining the element of carbon source with other formerly studied factors such as temperature and oxygen. Moreover, corn steep liquor showed promise for enhancement of growth in the oleaginous strains provided that carbon substrate is available.
Topics: Basidiomycota; Biofuels; Biomass; Carbon; Fermentation; Lipid Metabolism; Lipids; Nitrogen; Rhodotorula; Yeasts
PubMed: 34876116
DOI: 10.1186/s12934-021-01710-3 -
Marine Drugs Oct 2021Two known Polybrominated Diphenyl Ethers (PBDEs), 3,4,5-tribromo-2-(2',4'-dibromophenoxy)phenol () and 3,4,5,6-tetrabromo-2-(2',4'-dibromophenoxy)phenol (), were...
Two known Polybrominated Diphenyl Ethers (PBDEs), 3,4,5-tribromo-2-(2',4'-dibromophenoxy)phenol () and 3,4,5,6-tetrabromo-2-(2',4'-dibromophenoxy)phenol (), were isolated from the Indonesian marine sponge . The structure was confirmed using C chemical shift average deviation and was compared to the predicted structures and recorded chemical shifts in previous studies. We found a wide range of bioactivities from the organic crude extract, such as (1) a strong deterrence against the generalist pufferfish , (2) potent inhibition against environmental and human pathogenic bacterial and fungal strains, and (3) the inhibition of the Hepatitis C Virus (HCV). The addition of a bromine atom into the A-ring of compound resulted in higher fish feeding deterrence compared to compound . On the contrary, compound showed only more potent inhibition against the Gram-negative bacteria (MIC 2.1 μg/mL), while compound showed more powerful inhibition against the other human pathogenic bacteria and fungi. The first report of a chemical defense by compounds and against fish feeding and environmental relevant bacteria, especially pathogenic bacteria, might be one reason for the widespread occurrence of the shallow water sponge in Indonesia and the Indo-Pacific.
Topics: Animals; Antiviral Agents; Aquatic Organisms; Ecosystem; Halogenated Diphenyl Ethers; Hepacivirus; Indonesia; Microbial Sensitivity Tests; Porifera
PubMed: 34822482
DOI: 10.3390/md19110611 -
Frontiers in Microbiology 2021Diazinon is an organophosphorus pesticide widely used to control cabbage insects, cotton aphids and underground pests. The continuous application of diazinon in... (Review)
Review
Diazinon is an organophosphorus pesticide widely used to control cabbage insects, cotton aphids and underground pests. The continuous application of diazinon in agricultural activities has caused both ecological risk and biological hazards in the environment. Diazinon can be degraded via physical and chemical methods such as photocatalysis, adsorption and advanced oxidation. The microbial degradation of diazinon is found to be more effective than physicochemical methods for its complete clean-up from contaminated soil and water environments. The microbial strains belonging to sp., sp., , , , , and were found to be very promising for the ecofriendly removal of diazinon. The degradation pathways of diazinon and the fate of several metabolites were investigated. In addition, a variety of diazinon-degrading enzymes, such as hydrolase, acid phosphatase, laccase, cytochrome P450, and flavin monooxygenase were also discovered to play a crucial role in the biodegradation of diazinon. However, many unanswered questions still exist regarding the environmental fate and degradation mechanisms of this pesticide. The catalytic mechanisms responsible for enzymatic degradation remain unexplained, and ecotechnological techniques need to be applied to gain a comprehensive understanding of these issues. Hence, this review article provides in-depth information about the impact and toxicity of diazinon in living systems and discusses the developed ecotechnological remedial methods used for the effective biodegradation of diazinon in a contaminated environment.
PubMed: 34790174
DOI: 10.3389/fmicb.2021.717286